The Complexity of Gene Silencing

For this article, Jim Kling interviewed University of Edinburgh geneticist Adrian Bird and Paul A. Wade, assistant professor in the department of pathology and laboratory medicine at Emory University School of Medicine in Atlanta. Data from the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. H.-H. Ng, Y. Zhang, B. Hendrich, C.A. Johnson et al., "MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex," Nature Genetics, 23:58-61, September 1999 (Cited in 126 papers). P.A. Wade, A. Gegonne, P.L. Jones, E. Ballestar et al., "Mi-2 complex couples DNA methylation to chromatin remodelling and histone deacetylation," Nature Genetics, 23:62-6, September 1999 (Cited in 167 papers). Histone acetylation and DNA methylation are two important chemical changes involved in the intricate choreography of transcriptional regulation.1,2 Scientists know that they work in concert to turn genes on and off and regulate their expression. Researchers have found it difficult to draw up the playbill for the cast of characters performing these and other maneuvers. These two papers helped fill in some of the blanks by using converging methods to show the physical association between histone deacetylases and methyl-CpG binding proteins. Histones, proteins that form a cylindrical tuna-can-like structure, act as a scaffold around which DNA coils. Histone deacetylases, as their name implies, remove acetyl groups from the histone proteins. The resulting chemical changes induce structural realignments that could trigger or repress gene transcription. Meanwhile, DNA methylation leads to gene silencing. The fact that the two functions are wrapped in the same complex provides an intriguing mechanism for how genes get silenced. Courtesy of Adrian BirdAdrian Bird University of Edinburgh geneticist Adrian Bird and his team approached the problem from the perspective of methylated DNA, while Paul A. Wade, assistant professor in the department of pathology and laboratory medicine at the Emory University School of Medicine in Atlanta, and colleagues began with histone deacetyl-ase activity. Despite the different thought processes, the results of both efforts reached the same conclusion: binding to methylated DNA is directly linked to histone deacetylases that restructure the chromatin and dampen transcription. "In terms of functional outcome, the [studies] are exactly equivalent," says Wade. Leaving the Islands One interesting feature of the human genome is its "CpG islands," which are long stretches containing large numbers of cytosine-guanine dinucleotides. In the late 1980s, Bird's team noticed that some stretches of CpG islands, often near promoters, were unmethylated. They began to search human HeLa cells for proteins capable of binding preferentially to methylated DNA. "Most of the genome is heavily methylated, [so] how do some regions escape?" says Bird. "I hypothesized that there may be a protein that bound to non-methylated DNA to protect if from methylating enzymes. To our surprise, when we looked, nothing bound to the nonmethylated DNA, but something did bind strongly to the methylated DNA that we had in the experiment as a negative control.... So we forgot about what it takes to make a CpG island and focused on proteins that bound to methylated DNA instead." Bird's group isolated the methylated DNA-binding activity and found a complex they named MeCP, (now called MeCP1). MeCP1 caused transcriptional repression of a reporter gene, but purifying and identifying its components turned out to be a challenge, in part because of its large size, as large complexes are more difficult to purify, according to Bird. "We thought we could do it by DNA affinity, but every time we did that ... the act of binding to the DNA seemed to destroy the complex." Over the next decade, as biochemical techniques slowly caught up to the challenge of purifying the complex (which Wade's team accomplished and described in its paper), evidence was accumulating that histone deacetylases played a role in the chromatin complexes that in turn influence gene transcription. Focus on Mt. Everest Histone deacetylation caught the eye of Alan Wolffe, then at the National Institute of Child Health and Human Development. "We had some emerging data from studies of methylated DNA, and [the] finding was that micro-injected DNA templates that were methylated were profoundly repressed, and that repression required time for chromatin assembly," says Wade, who was a member of Wolffe's team at the time. "This repression was sensitive to [a histone deacetylase inhibitor]." Wade joined the team with an expertise in protein biochemistry, and was put to work developing an assay for isolating histone deacetylatases from Xenopus laevis (African frog) egg extracts. One histone deacetylase turned out to be far more abundant than the others. "We went for the biggest peak," he recalls. The histone deacetylase activity was linked to a complex with six subunits, and the smallest of them was MBD3, a protein that is known to bind to methylated DNA. The finding links a methylated DNA-binding activity to the histone deacetylase activity (also contained in the complex) that causes changes in transcription, providing an "attractive mechanism for explaining why DNA that is modified by methylation is so profoundly repressed," says Wade. It suggests that MBD3 binds methylated DNA, bringing with it a histone deacetylase that restructures the chromatin, leading to suppression of transcription. All in the Family Meanwhile, Bird's interest in MeCP1 continued. During efforts to purify MeCP1, his team isolated a related protein, MeCP2, which had similar properties. With its sequence in hand, Bird and coworkers began to search the sequences in public databases for matches. They found a family of proteins with a similar sequence in MeCP2's DNA-binding domain, which turned out to part of the same protein group that Wade had observed, the MBDs. One family member, MBD2, was well known to bind to methylated DNA in vitro and in vivo. Still focused on the MeCP1 complex, Bird's team used an anti-MBD2 antibody to demonstrate that MBD2 is present within it. The defining moment came when the anti-MBD2 antibody stripped the cell extract of its MeCP1 activity. "We knew the antibody was specific and it just took away all the activity. As far as I was concerned, that [action] was quite strong evidence that MBD2 was in this complex," says Bird. The team then assayed for histone deacetylase activity, and found that 27% of the cell extract's deacetylase potency had dropped out with the MBD2 and its antibody, suggesting that the enzyme responsible was linked to MBD2. That link was later confirmed when an antibody directed against one of the deacetylases also captured the MeCP1 complex and MBD2. "It was a bit of a breakthrough in helping to understand how DNA methylation (relates to) co-repressor complexes that change chromatin structure," says Bird. In the course of the research, Danny Reinberg of the Molecular Biology Program, Memorial Sloan-Kettering Cancer Center in New York City, was working on another HeLa-derived complex he called NuRD (nucleosome remodeling) complex. "But he didn't have a DNA binding factor," says Bird, who offered Reinberg's group access to MBD2. Reinberg's team went on to show that the complex contained MBD2.3 Both papers turned out to be an interesting response to an earlier paper that claimed MBD2 was a demethylase,4 but neither Bird's nor Wade's team could reproduce those results, and both came out in public and said so. "That's a fairly unusual thing, for research groups to go out on a limb and say they can't do something," says Bird. No one is quite sure what genes these complexes target, but Bird's team performed a knockout study of the mouse homolog of MBD2, which produced animals that seemed normal, except that they abandoned their offspring.5 "We now want to bridge the gap between DNA methylation and maternal behavior," says Bird. Wade is thinking along similar lines. "A large number of us are thinking about function.... There is a little evidence that some subunits of the complex are important for patterning or development in Caenorhabditis elegans; how it gets recruited, what it does to the local chromatin architecture is unknown. These are [questions] that will keep many of us busy, I would predict, for at least a decade." Jim Kling (jkling@nasw.org) is a freelance writer in Washington, DC. References 1. E. Russo, "Hot papers in acetylation," and "Hot papers in deacetylation," The Scientist, 13[5]:13, March 1, 1999. 2. B.A. Maher, "Researchers focus on histone code," The Scientist, 15[18]:15, Sept. 17, 2001. 3.Y. Zhang et al., "Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation," Genes & Development, 13:1924-35, 1999. (Cited in 117 papers). 4.S.K. Bhattacharya et al., "A mammalian protein with specific demethylase activity for mCpG DNA," Nature, 397:579-83, 1999. 5. B. Hendrich et al., "Closely related proteins MBD2 and MBD3 play distinctive but interacting roles in mouse development," Genes & Development, 15:710-23, March 15, 2001.

The Complexity of Gene Silencing

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